Expression of a Drosophila glutathione transferase in Arabidopsis confers the ability to detoxify the environmental pollutant, and explosive, 2,4,6‐trinitrotoluene

Abstract

Summary 26 The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental 27 pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale, and 28 inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up 29 approach. Here, we have characterised a Drosophila melanogaster (Meigen, 1830) 30 glutathione transferase (DmGSTE6) which has activity towards TNT. 31  Recombinantly-expressed, purified DmGSTE6 produces predominantly 2-32 glutathionyl-4,6-dinitrotoluene, and has a 2.5-fold higher V max , and 5-fold lower K m 33 than previously characterised TNT-active Arabidopsis thaliana (L. 2004), and many contaminated sites in Europe and Asia (Kalderis et al., 2011; Pichtel, 2012). 59)For example, the Werk Tanne former ammunition site in Germany, detonated in 1944, is 60 heavily contaminated with TNT (Eisentraeger et al., 2007). Increased environmental 61 awareness is now compelling governments to identify sites of explosives contamination and 62 put together remediation strategies (Lima et al., 2011). However, a major challenge to 63 cleaning-up these sites is the sheer scale and hazardous nature of many contaminated sites, 64 which rules-out many strategies such as excavation, land fill and off-site treatments, as 65 prohibitively expensive. Phytoremediation may be a viable alternative approach. 66TNT is not readily degraded in the environment due to the electron-withdrawing properties of 67 the three nitro groups of TNT which render the aromatic ring particularly resistant to 68 oxidative attack and ring cleavage (Qasim et al., 2009); the main route of aromatic 69 compounds by soil microbes. Instead microbial flora catalyse a series of reductive reactions, 70 producing predominantly hydroxylamino dinitrotoluene (HADNT) and amino dinitrotoluene 71 (ADNT) and further reduced derivatives (Rylott et al., 2011b). In plants, HADNT and ADNT 72 can be conjugated to sugars, for example, to glucose by UDP-glucosyltransferases (Gandia-73 Herrero et al., 2008), and it has recently been shown that glutathione transferases can 74 conjugate the TNT molecule directly (Gunning et al., 2014; Rylott et al., 2015). Two 75Arabidopsis thaliana (L.) Heynh (Arabidopsis) glutathione transferase (GST) genes, AtGST-76 U24 and AtGST-U25, are specifically upregulated in response to TNT exposure, and their 77 gene products catalyse the formation of three characterised TNT glutathionyl-products 78 (Gunning et al., 2014). The removal of a nitro group in one of the three products, 2-79 glutathionyl-4,6-dinitrotoluene, has the potential to be more amenable to subsequent 80 biodegradation in the environment, a property that could be applied in planta for the 81 5 detoxification of TNT in the field. Expression of AtGST-U24 and AtGST-U25 in Arabidopsis 82 conferred increased ability to take up and detoxify TNT; however, in the absence of TNT, 83 overexpression of these GSTs caused a reduction in plant biomass; an effect with deleterious 84 implications for xenobiotic detoxification...

Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants

“…GSTU45 from P. trichocarpa was previously found to have very modest catalytic activities with a variety of conventional GST substrates ( Musdal and Mannervik, 2015 ). Particular interest was directed to its potential activity with TNT in relation to phytoremediation ( Tzafestas et al , 2017 ). The present investigation was undertaken to find out to what extent the feeble activities of GSTU45 could be enhanced by protein engineering based on the infolog approach.…”

Right on target: using plants and microbes to remediate explosives

“…To enhance the production of conjugate 3 in planta a Drosophila melanogaster GST (dGST), that produces almost exclusively conjugate 3, was expressed in Arabidopsis. Although enhanced resistance to TNT was observed in the dGST plants, at higher TNT concentrations, detoxification was limited by GSH availability (Tzafestas et al 2017). Thus, increasing TNT phytodetoxification in plants might require correspondingly increasing GSH availability.…”

Creation of Environmentally Friendly Super “Dinitrotoluene Scavenger” Plants

“…Due to the higher costs associated with traditional methods of remediating nitroaromatic explosives‐contaminated sites, [ 13 ] alternative strategies such as bacterial or plant remediation, also known as bacterial mineralization or phytoremediation, have emerged. For example, phytoremediation has been studied for its ability to remediate 2,4‐DNT [ 14 ] and TNT, [ 15 , 16 ] which serve as intermediate and final products in the synthesis of nitroaromatic explosives, respectively. The microorganisms are primarily responsible for converting toxic materials into less‐toxic or innocuous materials.…”